Localization of the chemoreceptive properties of the surface membrane ofParamecium tetraurelia

Summary The plasma membrane ofParamecium tetraurelia comprises two morphologically distinct components; a membrane that encloses the cell body and a ciliary membrane. In order to investigate the relative contributions of the two membranes to attractant-induced membrane potential changes, cells were deciliated with ethanol and their subsequent responses to attractants examined.Deciliation did not significantly affect the magnitude of the hyperpolarizations evoked by acetic or lactic acids, and had no effect on the concentration dependence of responses to folic acid. We conclude that the components necessary for detection and response to attractants are not exclusive to the ciliary membrane ofP. tetraurelia. Deciliation ofParamecium concomitantly permits localized chemical stimuli to be applied directly to the cell surface in the absence of strong fluid currents that are generated by the activity of the locomotory organelles. By systematically applying K₂ folate to a number of sites on the cell surface, it has been possible to demonstrate an anterior-posterior gradient of chemosensitivity on the cell body ofP. tetraurelia.     

Primary ciliary dyskinesia relative protein ZMYND10 is involved in regulating ciliary function and intraflagellar transport in Paramecium tetraurelia

Cilia are highly conserved in most eukaryotes and are regarded as an important organelle for motility and sensation in various species. Cilia are microscopic, hair-like cytoskeletal structures that protrude from the cell surface. The major focus in studies of cilia has been concentrated on the ciliary dysfunction in vertebrates that causes multisymptomatic diseases, which together are referred to as ciliopathies. To date, the understanding of ciliopathies has largely depended on the study of ciliary structure and function in different animal models. Zinc finger MYND-type containing 10 (ZMYND10) is a ciliary protein that was recently found to be mutated in patients with primary ciliary dyskinesia (PCD). In Paramecium tetraurelia, we identified two ZMYND10 genes, arising from a whole-genome duplication. Using RNAi, we found that the depletion of ZMYND10 in P. tetraurelia causes severe ciliary defects, thus provoking swimming dysfunction and lethality. Moreover, we found that the absence of ZMYND10 caused the abnormal localization of the intraflagellar transport (IFT) protein IFT43 along cilia. These results suggest that ZMYND10 is involved in the regulation of ciliary function and IFT, which may contribute to the study of PCD pathogenesis.     

Roles of Adenylate Cyclases in Ciliary Responses of Paramecium to Mechanical Stimulation

Paramecium shows rapid forward swimming due to increased beat frequency of cilia in normal (forward swimming) direction in response to various kinds of stimuli applied to the cell surface that cause K⁺‐outflow accompanied by a membrane hyperpolarization. Some adenylate cyclases are known to be functional K⁺ channels in the membrane. Using gene‐specific knockdown methods, we examined nine paralogues of adenylate cyclases in P. tetraurelia to ascertain whether and how they are involved in the mechanical stimulus‐induced hyperpolarization‐coupled acceleration of forward swimming. Results demonstrated that knockdown of the adenylate cyclase 1 (ac1)‐gene and 2 (ac2)‐gene inhibited the acceleration of forward swimming in response to mechanical stimulation of the cell, whereas that spared the acceleration response to external application of 8‐Br‐cAMP and dilution of extracellular [K⁺] induced hyperpolarization. Electrophysiological examination of the knockdown cells revealed that the hyperpolarization‐activated inward K⁺ current is smaller than that of a normal cell. Our results suggest that AC1 and AC2 are involved in the mechanical stimulus‐induced acceleration of ciliary beat in Paramecium.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Adenylate cyclase in cilia from Paramecium: Localization and partial characterization

A particulate adenylate cyclase was identified in the excitable ciliary membrane from Paramecium tetraurelia. MnATP was preferentially used as substrate, the K_m was 67 μM, V_max was 1 nmol cAMP.min^?1.mg^?1, a marked temperature optimum of 37°C was observed. Adenylate cyclase was not inhibited by 100 μM EGTA or 100 μM La³⁺, whereas under these conditions guanylate cyclase activity was abolished. Fractionation of ciliary membrane vesicles by a Percoll density gradient yielded two vesicle populations with adenylate cyclase activity. In contrast, calmodulin/Ca-dependent guanylate cyclase was associated with vesicles of high buoyant density only.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.     

Behavioral Mutants in PARAMECIUM CAUDATUM

Mutants of Paramecium caudatum with abnormal swimming behavior or responses to cations were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. Some of the mutants, like pawn in P. tetraurelia, cannot swim backward and are called CNR. Seven independently obtained CNR mutants belonged to three complementation groups, designated as cnrA, cnrB and cnrC. Some characteristics of double homo- and heterozygotes were compared with single homo- and heterozygotes. Other behavioral mutants shown to have a genic basis included K⁺-sensitive, temperature-shock behavioral and slow swimmer. All those mutants except for slow swimmer had lesions in the membrane because Triton-extracted models of them show almost the same swimming behavior as wild type.     

Adenylate kinase in sea urchin embryonic cilia

Sea urchin embryos swim by ciliary movement. Hypertonic shock causes deciliation and loss of motility. Within 2-4 h, cilia regenerate and the embryos swim again. Regeneration of cilia occurs multiple times. The adenylate kinase (AK) activity of isolated cilia was studied. A 130-kDa Sp-AK isozyme, present in sperm flagella, is also present in embryonic cilia. AK activity is responsible for approximately 93% of nonmitochondrial ATP regeneration from ADP in embryonic cilia. This is unlike sea urchin sperm flagella, where approximately 31% of the nonmitochondrial ATP regeneration is from the 130-kDa Sp-AK isozyme and approximately 69% from the flagellar creatine kinase (Sp-CK). Embryos were deciliated 1-3 times and after a 2-h period of regeneration the major ciliary axonemal proteins such as the tubulins appeared constant in amount. However, a moderate decrease in ATPase activity, and a large decrease of total AK activity, were measured. The decrease in AK activity paralleled the decrease in embryo swimming velocity. Embryos were deciliated once and cilia regeneration followed for 4 h. ATPase activity recovered to control levels by 3 h, but AK activity and swimming velocity remained lower than in controls. Detergent solubility data and kinetic experiments indicate that, in addition to the 130-kDa Sp-AK, there is at least one additional AK isozyme in embryonic cilia. Analysis of the S. purpuratus genome indicates five AK isozymes in addition to the 130-kDa Sp-AK isozyme. Decreased swimming velocity of embryos with regenerated cilia suggests that regenerated cilia are not as functionally perfect as naturally grown cilia.     

Evidence for a correlation between swimming velocity and membrane fluidity of Tetrahymena cells

The influence of the physical state of the membrane on the swimming behaviour of Tetrahymena pyriformis was studied in cells with lipid-modified membranes. When the growth temperature of Tetrahymena cells was increased from 15°C to 34°C or decreased from 39°C to 15°C, their swimming velocity changed gradually in a similar to the adaptive change in membrane lipid composition. Therefore, such adaptive changes in swimming velocity were not observed during short exposures to a different environment. Tetrahymena cells adapted to 34°C swam at 570 μm/s. On incubation at 15°C these cells swam at 100 μm/s. When the temperature was increased to 34°C after a 90-min incubation at 15°C, the initial velocity was immediately recovered. On replacement of tetrahymanol with ergosterol, the swimming velocity of 34°C-grown cells decreased to 210 μm/s, and the cells ceased to move when the temperature was decreased to 15°C. To investigate the influence of the physical state of the membrane on the swimming velocity, total phospholipids were prepared from Tetrahymena cells grown under these different conditions. The fluidities of liposomes of these phospholipid were measured using stearate spin probe. The membrane fluidity of the cells cooled to 15°C increased gradually during incubation at 15°C. On the other hand, the fluidity of the heated cell decreased during incubation at 34°C. Replacement of tetrahymanol with ergosterol decreased the membrane fluidity markedly. Consequently, a good correlation was observed between swimming velocity and membrane fluidity; as the membrane fluidity increased, the swimming velocity increased linearly up to 600 μm/s. These results provide evidence for the regulation of the swimming behaviour by physical properties of the membrane.     

Paramecium GPI Proteins: Variability of Expression and Localization

In Paramecium primaurelia, the two major classes of cell surface proteins, the surface antigen (SAg) and the surface GPI proteins (SGPs), are linked to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. In the present study, we have characterized the expression of the SGPs in several geographical strains of P. primaurelia and P. tetraurelia at different temperatures, 23 °C and 32 °C. The identification of the expressed SGPs was performed on purified cilia, by establishing the SGP SDS-PAGE profiles under four different conditions: with or without their anchoring lipid, cleaved with a Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC), and either in a reduced or in an unreduced state. This screening revealed the existence of specific sets of ciliary SGPs, as a function of temperature and the geographical origin of the strains. The SGPs the most abundant at 23 °C and 32 °C displayed a rapid turnover. We also looked for the presence of PI-PLC releasable proteins in purified cortices. In addition to the SAg and SGPs, the cortical fraction was shown to contain other PI-PLC releasable proteins, not found in the ciliary fraction, thus localized exclusively in the interciliary region.     

A kinome of 2600 in the ciliate Paramecium tetraurelia

Protein kinases play a crucial role in the regulation of cellular processes. Most eukaryotes reserve about 2.5% of their genes for protein kinases. We analysed the genome of the single-celled ciliate Paramecium tetraurelia and identified 2606 kinases, about 6.6% of its genes, representing the largest kinome to date. A gene tree combined with human kinases revealed a massive expansion of the calcium calmodulin regulated subfamily, underlining the importance of calcium in the physiology of P. tetraurelia. The kinases are embedded in only 40 domain architectures, contrasting 134 in human. This might indicate different mechanisms to achieve target specificity.     

Biophysical effects of the natural product euplotin C on the <Emphasis Type="Italic">Paramecium</Emphasis> membrane

The effect of euplotin C—a cytotoxic secondary metabolite produced by the protist ciliate Euplotes crassus—on the voltage-dependent Ca²⁺ channel activity was studied in a single-celled system by analyzing the swimming behavior of Paramecium. When the intraciliary Ca²⁺ concentration associated with plasma membrane depolarization increases, a reversal in the direction of ciliary beating occurs, and consequently the swimming direction changes. The ciliary reversal duration is correlated with the amount of Ca²⁺ influx. The present study demonstrates that the duration of continuous ciliary reversal (CCR), triggered by high external KCl concentrations, is longer in euplotin C-treated cells. Using selective Ca²⁺ channel blockers, we demonstrate that euplotin C modulates Ca²⁺ channels similar to the T- and L-types that occur in mammalian cells. Indeed, the increase of CCR duration significantly decreased when flunarizine and nimodipine-verapamil blockers were employed. Membrane fluidity measurements using a fluorescent dye, 6-lauroyl-2-dimethylaminonaphtalene (laurdan), indicated that membranes in euplotin C-treated cells are more tightly packed and ordered than membranes in control cells. Our data suggest that euplotin C enhances backward swimming in our unicellular model system by interacting with the ciliary Ca²⁺ channel functions through the reduction of cell membrane fluidity.     

Protein differences in cilia of mechanoreceptor hair and gill cells of the scallop Mizuhopecten yessoensis

• 1.1. In search for mechanosensory molecules the composition of the ciliary proteins of mechanoreceptor hair cells of the abdominal organ and less mechanosensitive gill cells were compared electrophoretically.
• 2.2. The hair cells and gill cilia were very similar in their polypeptide sets but differed by contents of three axonemal polypeptides with molecular weights of 125, 149 and 300 kilodaltons (kDa) and one membrane polypeptide of 159 kDa.
• 3.3. The membrane polypeptide with a molecular weight of 159 kDa represented approximately 3% of the total ciliary protein of hair cells. There was only a trace of this polypeptide in gill cilia and their membrane fraction.
• 4.4. A peculiarity of ciliary membranes of the hair cells was a high content of the 159 kDa-polypeptide, which constituted more than 20% of the total protein of membrane fraction.

     

Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process.     

Divalent Cation-dependent ATPase Activities Associated with Cilia and Other Subcellular Fractions of Paramecium: An Electrophoretic Characterization on Triton-polyacrylamide Gels1

Several divalent cation-dependent ATP phosphohydrolases associated with cilia, ciliary axonemes, ciliary membranes, pellicles, trichocysts, nuclei, mitochondria, microsomes, and soluble peripheral cell surface fractions of Paramecium tetraurelia were resolved in this study. Fifteen different activity bands were detected in whole cell sonicates or subcellular fractions by Triton polyacrylamide gel electrophoresis and ATPase activity staining. The ciliary surface membrane contained two major ATPase activities that were distinct from the enzymes associated with all other cell fractions.     

Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase

Over a hundred proteins in eukaryotic cells carry a C-terminal CaaX box sequence, which targets them for posttranslational isoprenylation of the cysteine residue. This modification, catalyzed by either farnesyl or geranylgeranyl transferase, converts them into peripheral membrane proteins. Isoprenylation is usually followed by proteolytic cleavage of the aaX tripeptide and methylation of the carboxyl group of the newly exposed isoprenylcysteine. The C-terminal modification regulates the cellular localization and biological activity of isoprenylated proteins. We have established a strategy to produce and purify recombinant farnesylated guanylate-binding protein 1 (hGBP1), a dynamin-related large GTPase. Our system is based on the coexpression of hGBP1 with the two subunits of human farnesyltransferase in Escherichia coli and a chromatographic separation of farnesylated and unmodified protein. Farnesylated hGBP1 displays altered GTPase activity and is able to interact with liposomes in the activated state.     

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34⁺ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34⁺ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34⁺ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34⁺ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34⁺ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34⁺ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.     

In vitro and in vivo phosphorylation of polypeptides in plasma membrane and tonoplast-enriched fractions from barley roots

Phosphorylation of polypeptides in membrane fractions from barley (Hordeum vulgare L. cv CM 72) roots was compared in in vitro and in vivo assays to assess the potential role of protein kinases in modification of membrane transport. Membrane fractions enriched in endoplasmic reticulum, tonoplast, and plasma membrane were isolated using sucrose gradients and the membrane polypeptides separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis. When the membrane fractions were incubated with γ-[³²P]ATP, phosphorylation occurred almost exclusively in the plasma membrane fraction. Phosphorylation of a band at 38 kilodaltons increased as the concentration of Mg²⁺ was decreased from millimolar to micromolar levels. Phosphorylation of bands at 125, 86, 58, 46, and 28 kilodaltons required millimolar Mg²⁺ concentrations and was greatly enhanced by Ca²⁺. When roots of intact plants were labeled with [³²P]orthophosphate, polypeptides at approximately 135, 116, 90, 46 to 53, 32, 28, and 19 kilodaltons were labeled in the plasma membrane fraction and polypeptides at approximately 73, 66, and 48 kilodaltons were labeled in the tonoplast fraction. Treatment of the roots of intact plants with 150 millimolar NaCl resulted in increased phosphorylation of some polypeptides while treatment with 100 mm NaCl had no effect.